6520211Molecular Libraries Program, Specialized Chemistry Center, University of KansasKUA10019_BacCap_Cytotox2013710Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (4)Cytoxicity Assay Rationale and Summary: acquired urinary tract infections (UTIs). Over 100 million UTIs occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. Antimicrobial resistance among UPEC is on the rise, driving efforts to elucidate vulnerable targets in the molecular pathogenesis of infection. New insights into the roles of K capsules in UPEC virulence during UTI make capsules an attractive target. Uropathogenic Escherichia coli (UPEC) produces 80% of community-acquired urinary tract infections (UTI). UPEC is also a leading cause of nosocomial UTI, the most prevalent hospital acquired infection. Dissemination of UPEC from the lower urinary tract is associated with morbidity and mortality through infection of the kidneys, bloodstream, and central nervous system. In recent years, the treatment of outpatient and inpatient UTI has been severely compromised by the rising incidence of antibiotic-resistant UPEC.Investigators have found that encapsulation is an important UPEC virulence factor. The K1 capsule type is closely associated with pathogenic isolates; not only is it the leading type in UTI, but it also accounts for much of the extra-urinary tract complications. Animals studies of E. coli K1 sepsis demonstrated that injection of K1 capsule degrading enzyme abrogates infection. However, the enzyme treatment is immunogenic; accordingly, chemical inhibition may prove to be a superior approach.Of the different K types, the Group 2 and Group 3 capsules are most prevalent among UPEC isolates, with K1 and K5 being leading types. Although the capsules have different compositions, they are synthesized, assembled, and exported by functionally homologous factors, leading us to hypothesize that we can develop small molecular inhibitors of K-type encapsulation that target the most medically important K types. Furthermore, the medically important infectious agents Campylobacter jejuni, Hemophilus influenzae, Neisseria meningitides, and Salmonella typhimurium among others, use these homologues in the biogenesis of their capsules. By exploiting the properties of a K-type specific phage, we performed a small scale high-throughput screen of >2,100 molecules from the NCI that uncovered several promising inhibitors of K1 and K5 encapsulation. This assay will identify a larger number of inhibitors with different mechanisms of action from which we may determine the optimal targets for capsule biogenesis inhibition and develop analogues with pharmacologically optimized properties.Chemical modulators of K1 encapsulation might represent a new avenue to combat the catastrophic effects K1 diseases. To this end, this team has successfully developed a 1536-well high-throughput primary screen suitable for the discovery of novel capsule biogenesis inhibitors. This 384-well format counter-screen for hit toxicity in Bladder 5637 cells, along with the secondary assays for target validation will allow a prioritization of "hits" from the primary screen.Bladder 5637 Cell Cytotoxicity Dose Response Screen. The dose response testing established the compound cytotoxicity and subsequent release of LDH from non-viable cells and CC50 data necessary to determine the compounds advanced to secondary screens and target localization studies. Compounds were plated in 96-well microplates in quadruplicate . Bladder carcinoma 5637 cells were added to the compounds, and 72 hrs later LDH release was measured using p-Nitrophenyl phosphate tablets. Triton-X 100 (0.1%) was used as a positive cytotoxic control. The absorbance was then read at 405nm. Compound cytotoxicity and the 50% toxic concentration (CC50) was determined and compared to IC50 to calculate the therapeutic index. Cell viability was calculated relative to the mean of the cell control. Viability at each of eight tested concentrations ranging from 300 uM to 2.3 uM. Compounds that showed cell viability < 80% were considered Active (or Toxic). CC50 values were calculated using GraphPad Prism.The following tiered system has been implemented at The University of Kansas for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In confirmatory (dose response) screens active compounds are scored on a scale of 41-80 based on the CC50 result compounds that did not show activity were given the score 0. Active purified or synthesized compounds are scored on a scale of 81-100 based on the Average Active CC50 result with inactive compounds receiving the score of 0.Compounds that showed cell viability < 80% were considered Active (or Toxic). Compounds qualifying as Active in one replicate and Inactive in another are identified as Inconclusive.488966493020504769504358504543504675504768504349504538504767504733588321588322588386588399588395602408602414624055624056624061624060187764364106www.southernresearch.org21178466206578471CC50 Modifier42542CC50Cytotoxic concentration153% Cell Viability @ 300 uM115300514% Cell Viability @ 150 uM115150515% Cell Viability @ 75 uM11575516% Cell Viability @ 37.5 uM11537.5517% Cell Viability @ 18.75 uM11518.75518% Cell Viability @ 9.38 uM1159.38000011444092519% Cell Viability @ 4.69 uM1154.690000057220465110% Cell Viability @ 2.34 uM1152.3399999141693151321CR Plot Labels 1ConcentrationResponse01 R03 MH090791-012136964033121001=296.8323.9430.2544.4678.1786.5886.5989.31083.7http://www.scc.ku.edu2013271369640371201>2300384.2487581.6680.5777.5878.5977.51069.6http://www.scc.ku.edu2013271369640391101>2300310041005100610071008100910010100http://www.scc.ku.edu2013271369640471101>2300310041005100610071008100910010100http://www.scc.ku.edu2013271369640481101>2300310041005100610071008100910010100http://www.scc.ku.edu2013271369640491101>2300396.14100586.1610071008100910010100http://www.scc.ku.edu2013271369640501101>230031004100510061007100810091001097.6http://www.scc.ku.edu2013271369640511101>2300310041005100610071008100910010100http://www.scc.ku.edu20132713696405912801=2172320.1484.6510061007100810091001095.2http://www.scc.ku.edu201327